This invention relates to methods for the production of Pasteuria, or Pasteuria-like, bacteria. These bacteria are able to produce endospores that have the unique and useful property of being able to attach to, infect, grow in, re-sporulate in, and kill certain types of phytopathogenic nematodes and other soil-dwelling nematodes. These phytopathogenic nematodes come from the phylum Nematoda, within the orders Tylenchida and Dorylamida.
Plant parasitic nematodes inflict crop losses to world agriculture currently estimated to total US $80 billion. Preventing this damage represents a significant challenge. With the impending loss of the fumigant methyl bromide in 2005, there is insufficient time to develop and register new synthetic compounds for nematode control.
Phytopathogenic nematodes are particularly difficult to control because they are covered with a thick, impermeable cuticle, or outer covering, and have very few sensory neurons. Since many pest control compounds operate as neurotoxins, the low number of neurons exposed by phytopathogenic nematodes decreases the effective target area for nematicidal compounds and has resulted in the development of nematicidal compounds with exquisitely high neurotoxic properties. Furthermore, because the phytopathogenic nematodes are found in soil or plant roots, exposing the phytopathogenic nematodes to control agents also is difficult to achieve and puts the water table at risk of contamination from those toxic compounds. The use of nematicides based on neurotoxins has been demonstrated to contaminate both ground and surface water. Consequently, many of these compounds are being removed from the market for public health reasons.
Fumigation of soil prior to planting is a popular method for controlling nematodes. One of the most popular fumigants, methyl bromide, is slated for removal from use because of its ozone destroying properties. However, this practice of soil fumigation kills organisms in soil indiscriminately and runs the risk of eliminating beneficial microbes as well as disease organisms. The overall market for an effective nematicide with benign environmental effects is estimated to approach one billion dollars on a world-wide basis.
The genus Pasteuria has several members that parasitize plant-parasitic nematodes, and it has been recognized as a potential biological control agent. Although bacteria of the Pasteuria group have a recognized potential for use as biorational control agents against phytopathogenic nematodes, their widespread use in commercial agriculture will depend on the availability of reliable methods for the large-scale production of bacteria having specificity against the phytopathogenic nematodes of concern to farmers.
Pasteuria was first described in 1888 by Metchnikoff (Annales de l'Institut Pasteur 2:165–170) as a parasite of water fleas. Subsequently, Cobb described a Pasteuria infection of the nematode Dorylaimus bulbiferous (2nd ed. Hawaiian Sugar Planters Assoc., Expt. Sta. Div. Path. Physiol. Bull. 5:163–195, 1906). In the intervening years, Pasteuria infections of virtually every known nematode have been observed, and their potential for use in biological control of phytopathogenic nematodes has been noted (Chen and Dickson [1998] J. Nematology 30:313–340).
The life cycle of the bacteria begins when endospores bind to the cuticle of the nematodes in soil. P. penetrans proliferates within the nematode body and passes through several documented morphological phases, including mycelial structures and thalli, culminating in the development of endospores. Endospores are released when the nematode body lyses. Growth of the bacteria within the nematode body reduces or eliminates the production of eggs by the nematode, severely restricting the rate of nematode reproduction. Economic damage to the host crop normally is inflicted by the first generation progeny of nematodes and is prevented by Pasteuria through lowering the concentration of progeny nematodes in the plant root zone.
Most of the experimental work with the Pasteuria group of bacteria has used endospores produced in live nematodes, cultivated on whole plants in greenhouses where aseptic conditions do not prevail. In two exceptions, Verdejo et al. (Verdejo, S. and R. Mankau [1986] Journal of Nematology 18:635) have reported on the oligoxenic culture of Pasteuria penetrans in live Meloidogyne incognita on excised tomato root culture; and Reise et al. (Reise, R. W., et al. [1988] Abstracts of the 27th Annual Meeting Society of Nematologists, p. 75; Reise, R. W. et al. [1991] J. Nematol. 23:547–548) have studied factors in various tissue culture media affecting Pasteuria isolates from Heterodera glycines, Meloidogyne incognita, and Pratylenchus brachyurus. Their attempts are directed at a genuine in vitro cultivation of Pasteuria, which attempts fail on the basis of the fundamental criterion that a genuine in vitro cultivation of any prokaryotic organism must be marked by a continual survival and proliferation of the organisms, upon transfer to a fresh medium, at some definable growth rate that is characteristic of the genotype of the organism and the environmental conditions.
Previous attempts at culturing P. penetrans typically have relied on introducing vegetative cells from infected nematodes into selected culture media. These media included an extensive array of carbon and nitrogen sources, commercial growth media, extracts of other microorganisms or fungi, or tissue culture of nematode cells (Reise et al., 1988; Bishop and Ellar, 1991; Previc and Cox, 1992). However, sustained growth of Pasteuria was never observed in any of these media.
U.S. Pat. No. 5,094,954 describes an alternative method for producing endospores from Pasteuria by growing the bacteria on explanted nematode tissue. In the method of U.S. Pat. No. 5,094,954, the nematode tissue may be prepared, for example, by decapitating and decaudating nematodes, or by osmotic and/or enzymatic disruption of the nematode cuticle. The nematode tissue is explanted onto media which is designed to nourish the tissue and keep it in a metabolically active state. The tissue is then induced into growth and cell proliferation. Thus, this method does not rely on in vitro cultivation of the Pasteuria, but is directed at the production of Pasteuria endospores on explanted or cultured nematode tissue.
Thus, although Pasteuria was first reported as far back as 1888, all attempts to culture the microbe in vitro have failed to produce a viable means of producing endospores. Therefore, there remains in this art a great need for a method of producing Pasteuria by spore formation following true in vitro growth of the vegetative phase of Pasteuria on an artificial growth medium consisting of inexpensive, readily available materials. Such systems are not known at this time.